1. Field of the Invention
The present invention generally relates to bacterial polypeptide display libraries and methods of making and using thereof.
2. Description of the Related Art
Polypeptide display technologies have substantially impacted basic and applied research applications ranging from drug discovery to materials synthesis. See Clackson, T. and J. A. Wells (1994) Trends In Biotech. 12(5):173–184; and Shusta, E. V., et al. (1999) Curr. Opin. Biotechnol. 10(2):117–122; and Kodadek, T., (2001) Chem. Biol. 8(2):105–158; Lee, S. W., et al. (2002) Science 296 (5569):892–859; and Nixon, A. E. (2002) Curr. Pharm. Biotechnol. 3(1):1–12. The strength of these methods derives from the ability to generate libraries containing billions of diverse molecules using the biosynthetic machinery of the cell, and subsequently, to identify rare desired polypeptides using selection or high-throughput screening methods. Display libraries have been applied extensively to isolate and engineer peptides and antibodies for molecular recognition applications. In particular, display of peptides on the surface of filamentous bacteriophage, or phage display, has proven a versatile and effective methodology for the isolation of peptide ligands binding to a diverse range of targets. See Scott, J. K. and G. P. Smith (1990) Science 249(4967):386–904; Norris, J. D., et al. (1999) Science 285(5428):744–765; Arap, W., et al. (1998) Science 279(5349):377–806; and Whaley, S. R., et al. (2000) Nature 405(6787):665–668.
Polypeptide display systems include mRNA and ribosome display, eukaryotic virus display, and bacterial and yeast cell surface display. See Wilson, D. S., et al. 2001 PNAS USA 98(7):3750–3511; Muller, O. J., et al. (2003) Nat. Biotechnol. 3:312; Bupp, K. and M. J. Roth (2002) Mol. Ther. 5(3):329–3513; Georgiou, G., et al., (1997) Nat. Biotechnol. 15(1):29–3414; and Boder, E. T. and K. D. Wittrup (1997) Nature Biotech. 15(6):553–557. Surface display methods are attractive since they enable application of fluorescence-activated cell sorting (FACS) for library analysis and screening. See Daugherty, P. S., et al. (2000) J. Immuunol. Methods 243(1–2):211–2716; Georgiou, G. (2000) Adv. Protein Chem. 55:293–315; Daugherty, P. S., et al. (2000) PNAS USA 97(5):2029–3418; Olsen, M. J., et al. (2003) Methods Mol. Biol. 230:329–342; and Boder, E. T. et al. (2000) PNAS USA 97(20):10701–10705.
Phage display involves the localization of peptides as terminal fusions to the coat proteins, e.g., pIII, pIIV of bacteriophage particles. See Scott, J. K. and G. P. Smith (1990) Science 249(4967):386–390; and Lowman, H. B., et al. (1991) Biochem. 30(45):10832–10838. Generally, polypeptides with a specific function of binding are isolated by incubating with a target, washing away non-binding phage, eluting the bound phage, and then re-amplifying the phage population by infecting a fresh culture of bacteria. Unfortunately, phage display presents a few undesirable properties. See Zahn, G. (1999) Protein Eng. 12(12):1031–1034. For example, phage display is limited to about a few thousand copies of the displayed polypeptide per phage or less, thereby precluding the use of sensitive fluorescence-activated cell sorting (FACS) methodologies for isolating the desired sequences. Phage are also difficult to elute or recover from an immobilized target ligand, thereby resulting in clonal loss. Phage display also requires an infection step wherein viruses that do not bind and enter a cell are lost early in the process, thereby resulting in lower quality results overall, e.g., affinity of isolated binding molecules. Further, phage display selections are time consuming requiring typically about two to about three weeks for the isolation of phage display polypeptides that bind a given target.
Most notably, phage display requires that the investigator be familiar with routine phage manipulation methods including infections, phage amplifications, tittering, phage ELISA, and others. Second, phage display methods can lead to Darwinian outgrowth of particular clones owing to their relative infectivity, assembly efficiency, and toxicity to the host cell. Third, the rate at which desired binding clones can be enriched is slowed by relatively low enrichment ratios.
Other display formats and methodologies include mRNA display, ribosome or polysome display, eukaryotic virus display, and bacterial, yeast, and mammalian cell surface display. See Mattheakis, L. C., et al. (1994) PNAS USA 91(19): 9022–9026; Wilson, D. S., et al. (2001) PNAS USA 98(7):3750–3755; Shusta, E. V., et al. (1999) Curr. Opin. Biotech. 10(2):117–122; and Boder, E. T. and K. D. Wittrup (1997) Nature Biotech. 15(6):553–557. A variety of alternative display technologies have been developed and reported for display on the surface of a microogranism and pursued as a general strategy for isolating protein binding peptides without reported successes. See Maurer, J., et al. (1997) J. Bacteriol. 179(3):794–804; Samuelson, P., et al. (1995) J. Bacteriol. 177(6):1470–1476; Robert, A., et al. (1996) FEBS Letters 390(3): 327–333; Stathopoulos, C., et al. (1996) Appl. Microbiol. & Biotech. 45(1–2): 112–119; Georgiou, G., et al., (1996) Protein Engineering 9(2): 239–247; Haddad, D., et al., (1995) FEMS Immunol. & Medical Microbiol. 12(3–4):175–186; Pallesen, L., et al., (1995) Microbiol. 141(Pt 11): 2839–2848, Xu, Z. and S. Y. Lee (1999) Appl. Environ. Microbiol. 65(11):5142–5147; Wernerus, H. and S. Stahl (2002) FEMS Microbiol. Lett. 212(1): 47–54; and Westerlund-Wikstrom, B. (2000) Int. J. Med. Microbiol. 290(3):223–230. Some of these prior art display systems have been tested for library screening without success for isolation of high affinity protein binding peptides. See Brown, S. (1992) PNAS USA 89(18):8651–8655; Lang, H., et al. (2000) Eur. J. Biochem. 267(1):163–170; Klemm, P. and M. A. Schembri (2000) Int. J. Med. Microbiol. 290(3):215–221; Klemm, P. and M. A. Schembri (2000) Microbiol. 146(Pt 12):3025–3032; Kjaergaard, K., et al. (2000) Appl. Environ. Microbiol. 66(1):10–14; Schembri, M. A., (1999) FEMS Microbiol. Lett. 170(2):363–371; Benhar, I., et al. (2000) J. Mol. Biol. 301(4):893–904; and Lang, H., et al. (2000) Adv. Exp. Med. Biol. 485:133–136.
Prior art expression vectors for polypeptide display libraries using host cells suffer from a variety of problems. The problems of the prior art methods include (1) only small peptides may be expressed, (2) large libraries cannot be selected, (3) the polypeptides are not expressed on the outer membrane surface, but are instead expressed in the periplasmic space between the inner and the outer membranes, (4) polypeptides that are displayed on the outer membrane surface do not properly bind or interact with large molecules and certain targets, and (5) analyzing expression on fimbrial or flagella results in loss of some desired polypeptides due to mechanical shearing.
Protein display on the surface of bacterial cells holds the potential to simplify and accelerate the process of ligand isolation since experimental procedures with bacteria are efficient and screening can be performed using FACS. See Daugherty, P. S., et al. (2000) J. Immunol. Methods 243(1–2):211–2720; Brown, S. (1992) PNAS USA 89(18):8651–8521; and Francisco, J. A., et al. (1993) PNAS USA 90(22):10444–10448; Taschner, S., et al. (2002) Biochem. J. 367(Pt 2):393–402; Etz, H., et al. (2001) J. Bacteriol. 183(23):6924–6935; and Camaj, P., et al. (2001) Biol. Chem. 382(12):1669–1677. Though several different bacterial display systems have been reported, their usefulness has been restricted by technical limitations including accessibility on the cell surface, inability to display highly diverse sequences, and adverse effects on cell growth and viability. See Francisco, J. A., et al. (1993) PNAS USA 90(22):10444–10822; Lu, Z., et al. (1995) Biotechnology (NY) 13(4):366–7223; Klemm, P. and M. A. Schembri, (2000) Microbiology 146(Pt 12):3025–3224; Christmann, A., et al. (999) Protein Eng. 12(9):797–80625; Lee, S. Y., et al. (2003) Trends Biotechnol. 21(1):45–52; Lu, Z., et al. (1995) Biotechnology (NY) 13(4):366–7225; Lee, S. Y., et al. (2003) Trends Biotechnol. 21(1):45–5226; Camaj, P., et al. (2001) Biol. Chem. 382(12):1669–1677; and Schembri, M. A., et al. (2000) Infect. Immun. 68(5):2638–2646.
Consequently, these techniques do not enable isolation of high affinity peptide ligands. Additionally, these techniques do not provide peptide exposure on the cell surface suitable for binding to analytes including antibodies, proteins, viruses, cells, macromolecules. Thus, these display formats are not compatible with certain isolation methods, since the peptides produced do not bind to large molecules and other surfaces, e.g., magnetic particles. The prior art process also reduces cell viability and alters membrane permeability, thereby reducing process efficiency. Thus far, routine isolation of high affinity peptide ligands for arbitrary protein targets has not been demonstrated. See Camaj, P., et al., (2001) Biol. Chem. 382(12):1669–7727; and Tripp, B. C., et al., (2001) Protein Eng. 14(5):367–377; Lang, H., et al. (2000) Eur. J. Biochem. 267(1):163–170; Lang, H., et al. (2000) Adv. Exp. Med. Biol. 485:133–136; Klemm, P. and M. A. Schembri (2000) Int. J. Med. Microbiol. 290(3): 215–221; Klemm, P. and M. A. Schembri (2000) Microbiol. 146(Pt 12):3025–302; Kjaergaard, K., et al. (2000) Appl. Environ. Microbiol. 66(1):10–14; Schembri, M. A., et al. (1999) FEMS Microbiol. Lett. 170(2):363–371; Benhar, I., et al. (2000) Mol. Biol. 301(4):893–904; Kjaergaard, K., et al. (2001) Appl. Environ. Microbiol. 67(12):5467–5473; and Lang, H., et al. (2000) Exp. Med. Biol. 485:133–136.
Also, polypeptides in the prior art are most often displayed on cell surfaces either as insertional fusions or “sandwich fusions” into outer membrane or extracellular appendage, e.g., fimbria, flagella proteins, or less frequently, as fusions to truncated or hybrid proteins thought to be localized on the cell surface. See Pallesen, L., et al. (1995) Microbiol. 141(Pt 11):2839–48; and Etz, H., et al. (2001) J. Bacteriol. 183(23):6924–6935. Examples of the latter include the LppOmpA system and the ice nucleation protein (InP). See Georgiou, G., et al. (1997) Nat. Biotechnol. 15(1):29–34. The outer membrane proteins OmpA, OmpC, OmpF, FhuA, and LamB, have enabled the display of polypeptides as relative short insertional fusions into OMP loops exposed on the extracellular side of the outer membrane. See Xu, Z. and S. Y. Lee (1999) Appl. Environ. Microbiol. 65(11):5142–5147; Taschner, S., et al. (2002) Biochem. J. 367(Pt 2):393–402.
However, the C and N-termini of these “carrier” proteins are not naturally located on the cell surface which precludes the ability to display polypeptides as terminal fusions.
As a result, proteins which are not capable of folding in the insertional fusion context, when their C and N termini are fused to the “carrier” protein sequence, as well as those for which the C and N termini are physically separated in space, e.g., single chain Fv antibody fragments, cannot be displayed effectively as insertions. Similarly, the restriction to the use of insertional fusions, interferes with the display of a large number of proteins encoded by cDNA libraries on the cell surface.
Prior art methods have attempted to address the problems of insertional fusion displays by truncating outer membrane protein sequences such that the resulting new termini might be displayed on the cell surface. See Lee, et al. (2003) Trends in Biotech. 23(1):45–52; Georgiou, et al. (1997) Nat. Biotech. 15(1):29–34. These prior art approaches were used to create the LppOmpA system which allows for the targeting of peptides and polypeptides to the outer membrane of bacteria. See Francisco, et al. (1992) PNAS USA 89(7):2913. For example, expression vectors for which use LppOmpA′, araBAD promoter, chloramphenicol resistance, and a p15A origin (LppOmpA expression vector). See Daugherty et al. (1999) Protein Engineer. 12(7):613–621. The LppOmpA expression vector encodes a fusion protein that results in a truncation of the OmpA protein at amino acid residue 159. Unfortunately, the performance of LppOmpA expression vector as a general process for isolating and expressing polypeptides from large libraries is significantly restricted by i) the reduced structural stability of the modified OmpA protein, ii) intolerance to expression at high temperatures, iii) reduced viability, and iv) most importantly, its inability to display polypeptides on the cell surface in a manner compatible with binding to large proteins without compromising viability and/or growth rate See Christman, A. et al., 1999. Prot. Eng. 12 (9):797.
In addition, expression vectors in the prior art are problematic because (1) the polypeptides produced by the expression vectors are not capable of binding externally added proteins, cells, or surfaces to the host cells, (2) the expression vectors does not allow surface presentation of large polypeptides, and (3) the expressed polypeptides are only expressed in the periplasmic region (between the inner and outer membrane) and not on the outer surface of the host cell, and therefore any expressed protein can only interact with small molecules that pass through the outer membrane and into the periplasmic space. These problems have prevented the application of this technology as a general process for isolating high affinity binding polypeptides. See e.g., Stathopoulos, C. (1996) Applied Microbiol. Biotech. 45 (1–2) 112. Earhart C F. (2000) Methods Enzymol. (326):506–16; Francisco, J. (1994) Annal. NY Acad. Sci. 745:372; and Bessette, P. H., et al. (2004) Prot. Eng. (In Press).
Thus, a need exists for a more robust display methodology which requires minimal technical expertise, is less labor intensive, and speeds the process of ligand isolation from weeks to days as compared to the prior art methods.